Exploration of morpholine-thiophene hybrid thiosemicarbazones for the treatment of ureolytic bacterial infections via targeting urease enzyme: Synthesis, biochemical screening and computational analysis

An important component of the pathogenicity of potentially pathogenic bacteria in humans is the urease enzyme. In order to avoid the detrimental impact of ureolytic bacterial infections, the inhibition of urease enzyme appears to be an appealing approach. Therefore, in the current study, morpholine-thiophene hybrid thiosemicarbazone derivatives (5a-i) were designed, synthesized and characterized through FTIR, 1H NMR, 13C NMR spectroscopy and mass spectrometry. A range of substituents including electron-rich, electron-deficient and inductively electron-withdrawing groups on the thiophene ring was successfully tolerated. The synthesized derivatives were evaluated in vitro for their potential to inhibit urease enzyme using the indophenol method. The majority of compounds were noticeably more potent than the conventional inhibitor, thiourea. The lead inhibitor, 2-(1-(5-chlorothiophen-2-yl)ethylidene)-N-(2-morpholinoethyl)hydrazinecarbothioamide (5g) inhibited the urease in an uncompetitive manner with an IC50 value of 3.80 ± 1.9 µM. The findings of the docking studies demonstrated that compound 5g has a strong affinity for the urease active site. Significant docking scores and efficient binding free energies were displayed by the lead inhibitor. Finally, the ADME properties of lead inhibitor (5g) suggested the druglikeness behavior with zero violation.

In parallel, morpholine heterocycle has been ranked as an important pharmacophore encountered in numerous drugs used for the treatment of bacterial infections and other diseases (Bektaş et al., 2013;Bektaş et al., 2017;Kumari and Singh, 2020;Sıcak et al., 2023).The incorporation of morpholine ring into various drug molecules leads to enhanced potency through the formation of molecular interactions with the target protein or by modulating the pharmacokinetic profile.The World Drug Index has declared more than 100 drugs featuring morpholine ring (Kumari and Singh, 2020).Thiophene containing compounds, on the other hand, have also been reported to exhibit potent inhibitory efficacy against urease (Khan et al., 2010;Noreen et al., 2017;Akyüz et al., 2022;Güven et al., 2023).Both morpholine and thiophene rings have been precedented in literature showing significant urease inhibitory efficacy (Khan et al., 2010;Bektaş et al., 2013;Bektaş et al., 2017;Rego et al., 2018b;Akyüz et al., 2022).Similarly, thiosemicarbazones bearing diverse structural features have also been documented as potent anti-urease agents (Islam et al., 2019;Islam et al., 2023).In view of the anti-urease potential of all three distinct pharmacophores, we aimed to explore their combined urease inhibitory effect as a single entity to successfully deliver the lead drug candidates (Figure 1).Therefore, the structural optimization led us to design and synthesize a concise series of morpholine-and thiophene-containing thiosemicarbazone conjugates which were evaluated against urease enzyme.The obtained in vitro inhibitory data was reinforced with the molecular docking analysis elaborating the ligand-enzyme interaction within the active site of urease enzyme.Molecular dynamics simulations as well as ADMET properties were also computed.
The FTIR spectra of the synthesized thiosemicarbazones displayed N-H stretching bands at 3,345-3,312 cm −1 , C=N around 1,530-1,506 cm −1 and C=S stretching vibrations at 1,142-1,102 cm −1 . 1 H NMR spectra exhibited two peaks concerning the secondary thioamide protons referring to = N-NH and C-NH.The = N-NH signal appeared as a singlet peak downfield between 10.45 and 11.57ppm.The C-NH proton splits into a triplet due to its spin-spin coupling with the neighbouring methylene protons and was observed around 8.15 ppm.Hydrogens of morpholine ring gave two sets of triplets near 2.44 ppm and 3.60 ppm, the later signal though merged with the peak of the methylene in most of the compounds.The thiophene ring protons resonated between 6.78 and 8.09 ppm depending upon the substituent on the ring. 13C NMR spectral data endorsed the structures of the synthesized compounds where C=S functional group appeared at 178 ppm.Signals of morpholine ring carbon atoms resonated at 53.5 and 66.7 ppm while resonances for linear methylene carbon atoms were observed at 40.4 and 56.5 ppm.Thiophene ring carbon atoms resonated between 108.9 and 178.2 ppm.Likewise, the elemental analyses and mass spectral data of all the derivatives (5a-i) were in agreement with the anticipated structures.

In vitro urease inhibition and structureactivity relationship analysis
The synthesized morpholine-thiophene hybrid thiosemicarbazones (5a-i) were tested to determine their urease inhibitory effect.In comparison to the standard inhibitor, thiourea, which showed inhibitory activity with an IC 50 value of 22.31 ± 0.03 µM, these compounds were discovered to be effective inhibitors of the urease demonstrating outstanding inhibition with IC 50 in the range of 3.80-5.77µM.The inhibitory results of the tested compounds are shown in Table 1.
As depicted in Table 1, a varied degree of inhibitory potential was observed for tested compounds (5a-i).All the compounds showed remarkable inhibitory potential identifying compound 5g as the lead inhibitor exhibiting strong inhibitory potential with an IC 50 value of 3.80 ± 1.9 µM, more than the standard inhibitor.
In vitro analysis of compounds against urease showed that compound 5a ((E)-N-(2-morpholinoethyl)-2-(thiophen-2ylmethylene)hydrazinecarbothioamide) exhibited remarkable inhibitory potential with an IC 50 value of 4.94 ± 2.7 µM, 4.5-fold strong inhibitory potential than thiourea.Similar inhibition potential was observed for compound 5b where an additional methyl group was present at the imine moiety.Compound 5b demonstrated an IC 50 value of 4.96 ± 3.0 µM.However, the addition of methyl substituent at the thiophene ring on position 4 (5c) showed inhibitory potential with an IC 50 value of 4.00 ± 2.4 µM.Switching the position of methyl substituent at thiophene ring from 4 to 5 (5d) exhibited slightly lower inhibitory potential than 5c but the strength was still 4.8-fold better than thiourea.Furthermore, the replacement of methyl substituent with a chloro group at thiophene ring (position 3) demonstrated similar inhibition results with an IC 50 value of 4.81 ± 1.5 µM as shown by compound 5e.In compound 5f, the replacement of chloro with bromo demonstrated the least potential among all compounds (5a-i) with an IC 50 value of 5.77 ± 0.7 µM.However, the inhibitory strength was still stronger than thiourea.Compound 5g exhibited inhibitory potential with the lowest IC 50 value.The presence of the chloro group at the fifth position of thiophene ring demonstrated the highest inhibitory strength with an IC 50 value of 3.80 ± 1.9 µM.The presence of other electron-withdrawing groups (Br, NO 2 ) at the same position also showed remarkable inhibition of urease with IC 50 values of 3.98 ± 2.2 and 3.90 ± 2.7 µM, respectively, as depicted by compounds 5h and 5i.In general, all the tested compounds exhibited several folds higher inhibitory power compared to thiourea, standard inhibitor.Although a varied degree of inhibition was noticed among the tested series of thiosemicarbazones, the extent of biological efficacy was found least dependent on the type of substituent on thiophene ring.Therefore, considering the incorporation of further ring variations as well as changing the ethylmorpholine unit on the left hand side of the synthesized molecules could exert beneficial role in designing new families of thiosemicarbazones for potent urease inhibitory properties.

Kinetics analysis
The kinetics experiments of the most potent inhibitor 5g having the least IC 50 value were performed and the results obtained were utilized to construct Lineweaver-Burk plot using GraphPad Prism version 10.2.1.These experiments helped in the assessment of K m and V max values to predict the mechanism of urease inhibition by plotting reciprocal of reaction rate 1/V (y-axis) against reciprocal of substrate concentrations 1/[S] (x-axis).As illustrated in Figure 2, compound 5g inhibits urease in an uncompetitive manner as all the slopes (K m /V max ) are parallel to each other at different inhibitor concentrations.These results indicate that both the V max and K m decrease as the concentration of inhibitor increases.

Docking analysis
The lead compound exhibiting the best inhibitory potential with low IC 50 value was docked within the jack bean urease active site.Similar to literature examples (Elbastawesy et al., 2021;Hina et al., 2023), PDB ID: 3LA4 was selected as a computational urease model to perform the docking studies for compounds investigated in this research.Compound 5g demonstrated binding affinity in micromolar range along with various interactions with the active site amino acid residues, as shown in Table 2.
The analysis of intermolecular interactions of potent inhibitor 5g within the binding pocket of urease revealed that several amino acids including Leu13, Ala16, Tyr32, Ala37, Lys709, Phe712, Glu742, Val744 and Leu839 are significantly involved in the formation of key contacts.Compound 5g showed conventional hydrogen bonding with Tyr32, Glu742 and Lys709 present at a distance of 3.08, 2.26 and 2.88 A, respectively.However, carbon

Compound
Structure Urease inhibition [a] IC 50 ± SEM µM All experiments were performed in triplicate.

FIGURE 2
The Lineweaver-Burk plot of 5g at various substrate (urea) and inhibitor concentrations showing uncompetitive inhibition as indicated by parallel slopes.
The other compounds were also docked in the same active site, and the sequence of the decrease in estimated affinity (5g > 5i > 5h > 5c > 5d> 5e > 5a>5b > 5f > positive control) verifies the IC 50 values obtained in an in vitro assay against urease (Supplementary Figure S1).
The conventional inhibitor (thiourea) showed only three interactions (hydrogen bonds) with two amino acid residues (Ser421 and Thr715) when docked in the same binding pocket of urease.Therefore, it depicts more IC 50 value as compared to morpholine-thiophene hybrid thiosemicarbazone derivatives.Moreover, all the synthesized morpholine-thiophene hybrid thiosemicarbazones (5a-i) showed maximum intermolecular interactions with the active site residues of urease.That is why, their IC 50 values have no major differences.The thiophene ring as the main attachment with various substituents interacts with urease Illustration of 3D (A) and 2D (B) interactions of compound 5g with urease.
The presence of methyl substituent on the thiophene ring (5c and 5d) facilitates the formation of alkyl interaction.On the other hand, the bromo substituent on thiophene ring in 5f and 5h participates in the formation of alkyl interaction with amino acid residues of urease.In addition, nitro substitution on thiophene ring enables π-cation, π-anion and attractive charges interactions.The presence of chloro group as a substituent on the thiophene ring in 5e and 5g has not shown any interaction with the amino acid residues of the binding site.Apart from these, hydrogen bond interactions were observed as the common contacts within all compounds docked with the binding site residues of urease, but the number of these interactions varied as 5g showed 7 H-bond interactions.The presence of more hydrogen bond interactions makes 5g the most potent and lead inhibitor showing the lowest IC 50 value of 3.80 ± 1.9 µM.
These findings also correlate with the literature precedent (Hassan and Švajdlenka, 2017) where hydrogen bonding involving amino acid residues Tyr32 and Lys709 are mainly responsible for the anti-urease activity.Another similar report (Kataria and Khatkar, 2019) also revealed that compounds having the best antibacterial activity develop key interactions with the same amino acids (Val744, Lys709, Try32, Ala16, Ala37, Glu742, Leu839, and Leu13) as mentioned in Figure 3.However, the computational analysis of standard inhibitor (thiourea) does not match with the literature example (Saeed et al., 2013).Regardless of this study, several other reports showed the inhibition of urease by various compounds thus making crucial interactions with the same amino acids (Hassan and Švajdlenka, 2017;Kataria and Khatkar, 2019;Hina et al., 2023), as mentioned in Figure 3.These results showed that compound 5g has the potential to exhibit antibacterial activity by the inhibition of urease.

SeeSAR visual drug design
LeadIT software was used to evaluate the Hydrogen bond and DEhydration energy (HYDE) of potent inhibitor 5g.HYDE studies showed that the lead inhibitor (5g) has the highest affinity towards urease.Figure 4 demonstrates the HYDE of atoms of compound 5g indicating their involvement in the binding affinity.The lower the HYDE of an atom, the higher is its involvement in the estimated affinity.

ADMET properties
The ADMET properties of compound 5g were determined via calculating various parameters, including physicochemical properties, lipophilicity, water solubility, pharmacokinetics, druglikeness, and medicinal chemistry.These properties determine the likelihood of a drug.In our case, these properties indicated that compound 5g is the lead inhibitor with 0 violation, as shown in Table 3.The pharmacokinetic properties and the lowest IC 50 value support compound 5g to serve as a lead inhibitor.Furthermore, the comparative analysis of pharmacokinetic parameters of 5g from four different software (SwissADME, pkCSM, preADMET and vNN-ADMET) is shown in Table 4 (Dulsat et al., 2023).These results interpreted that compound 5g can be absorbed from gastrointestinal tract but unable to cross the barrier between blood and brain tissues.In addition, compound does not inhibit the P-glycoprotein (P-gp) but may act as a substrate of P-gp.The in depth toxicological analysis of 5g using admetSAR was also obtained in the form of probability score (Table 5).The prediction having probability of 0.7 or higher are more significant and included in the analysis of 5g.According to this, 5g exhibits non-carcinogenic and nonnephrotoxic character, whereas, it can induce respiratory and reproductive toxicity.Moreover, the compound does not cause any irritation to eyes or integumentary lining.Biotransformation 3.0 showed the reactive potential of 5g with bacterial enzyme UDP-glucuronosyltransferase (Table 6).This enzyme can biotransform compound 5g into another compound via N-glucuronidation of tertiary aliphatic amine.The same software was used to access the abiotic transformation of 5g and it was disclosed that compound could undergo reduction, ozonation and chlorination under abiotic conditions.Xenosite analysis interpreted that 5g can undergo epoxidation, N-dealkylation, and have reactive site to bind with the DNA (Table 6).The predictions of Xenosite are scored on the basis of color such as red color scored for 1, yellow between 1 and 0.5, green color for 0.5, blue between 0.5 and zero and white for zero.The higher the score, the greater will be the chance of that site to be reactive (Dang et al., 2020).

Molecular dynamics simulation
The molecular dynamics simulation was performed by using an iMOD server.This online server is used for the estimation of the stability of the ligand-protein complex.The results of molecular dynamics simulation of 5g are shown in Figure 5.The lower peaks are associated with lower deformability, while higher peaks represent higher deformability, as shown in Figure 5A.However, the eigenvalue represents the energy required for the deformation of the structure with an inverse relation, as shown in Figure 5B.Moreover, three different colors represent the covariance, such as correlated as red, noncorrelated as blue, and anti-correlated as white (Figure 5C). Figure 5D represents the elastic model network that is associated with spring formation.The gray color indicates the extent of spring formation, and most of the atoms are forming spring.
3 Materials and methods

Chemicals and instrumentation
The chemicals and solvents used to perform synthetic chemistry were of analytical grade and obtained from local dealers of Merck, Fisher and Fluka.Thin layer chromatography was carried out on aluminum plates coated with silica gel 60 F 254 (Merck) in an appropriate eluting system and UV lamp was employed for visualization of spots.Melting points were recorded in open capillaries on Gallenkamp melting point apparatus and are uncorrected. 1H NMR spectra were recorded in DMSO-d 6 on Bruker Avance NMR spectrometer at 300 MHz while 13 C NMR at 75 MHz.Chemical shifts are reported as δ values in parts per million (ppm) compared to TMS as internal standard or the residual deuterated solvent used.Coupling constant (J) is given in Hertz.FTIR spectra (neat) were recorded on Bruker FTIR spectrophotometer.Mass spectra were obtained using LCMS 6495c Agilent whereas elemental analysis was attained on LECO 630-200-200 TRUSPEC CHNS micro analyzer and the experimental values are within ±0.4% of the calculated values.
Synthesis of compounds 2 and 3 was achieved by following the reported synthetic procedures (Mandapati et al., 2017;He et al., 2019).

General procedure for the synthesis of thiosemicarbazones (5a-i)
To a stirred solution of N-(2-morpholinoethyl) hydrazinecarbothioamide 3 (1 mmol) in absolute ethanol (20 mL) was added thiophene-2-carbaldehyde (or un/substituted acetylthiophene) 4 (1 mmol) and two to three drops of glacial acetic acid.The reaction mixture was refluxed for 10-12 h and then allowed to stand for 48 h in a refrigerator.The precipitated crude product was filtered and recrystallized from methanol to give thiosemicarbazones (5a-i).

Urease inhibition assay
The indophenol method with a little modification was used to determine the urease inhibitory activity of synthetic compounds (5a-i) (Weatherburn, 1967;Yaseen et al., 2016).Thiourea was used as a standard inhibitor.The assay mixture was comprised of 40 μL of buffer (100 mmol/L urea, 0.01 mol/L K 2 HPO 4 , 1 mmol/L EDTA and 0.01 mol/L LiCl 2 , pH 8.2), 10 μL of jack bean urease (5 U/mL) and 10 μL of test compound (1 mM).The reaction mixture was incubated for 30 min with 10 μL urea (1 mM) at 37 °C in 96-well plate.The phenol reagent (40 μL, 1% w/v phenol, 0.005% w/v sodium nitroprusside) and alkali reagent (40 μL, 0.5% w/v NaOH, 0.1% active chloride NaOCl) were added to each well and after 10 min of incubation at 37 °C, the absorbance was measured at 630 nm using a microplate reader (Bio-Tek ELx 800 ™ , Instruments, Inc.United States).The experiments to determine the inhibitory activity of compounds were performed in triplicates.The percentage inhibition was calculated by using the formula given below.

Inhibition %
( ) 100 -Absorbance of the test compound Absorbance of the control] × 100 Compounds showing >50% inhibition against urease were further evaluated for the determination of IC 50 values at various concentrations (0.1, 0.3, 1, 3, 10, 30, 100, 300, 1,000 μM) against jack bean urease.The experiments were performed in triplicate.IC 50 values were calculated by using the non-linear curve fitting program PRISM 5.0 (GraphPad, San Diego, California, United States) to analyze the results.

Kinetics analysis
The most potent inhibitor 5g was examined for its mechanism of inhibition against urease by Michaelis-Menten kinetics.The rate of enzyme inhibition was accessed across numerous concentrations of substrate (25, 50, 100, 150 mM) and compound 5g (0, 1.9, 3.8, 5.7 μM) against urease.Afterwards, the data was plotted to obtain the Lineweaver-Burk plot by using GraphPad Prism version 10.2.1.

In silico investigation 3.5.1 Docking studies
For the molecular docking studies, the crystallographic structure of jack bean urease (PDB ID: 3LA4) was retrieved from the RSCB PDB database (Balasubramanian and Ponnuraj, 2010).The structures of compound and enzyme were prepared by protonation with the Protonate3D (Labute, 2007) algorithm implemented in MOE molecular modeling tool (http://www.chemcomp.com/MOEMolecular_Operating_Environment.htm).
However, the calculations for the docking investigations were performed using LeadIT from BioSolveIT, GmbH, Germany (www.biosolveit.de/LeadIT).The FlexX application of LeadIT was used for the docking of compounds.Based on the binding free energies, the conformation of the ligand-receptor complex was determined.No modifications were made to the default docking parameters (www.biosolveit.de/LeadIT).The highest affinity for interfacing with the receptor and the most stable poses were determined to possess the lowest free binding energies.Complexes were visualized using Discovery Studio Visualizer v4.

ADMET analysis
An online platform (SwissADME; http://www.swissadme.ch/index.php) was used to determine the pharmacokinetic properties of potent inhibitor 5g.Several properties such as physical and chemical characteristics, bioavailability, solubility, druglikeness, lipophilicity, pharmacokinetics, and medicinal chemistry of compound 5g were determined to represent the identified inhibitor as a safer therapeutic agent (Daina et al., 2017).The detailed toxicity analysis was conducted via another user-friendly interface admetSAR (http:// lmmd.ecust.edu.cn/admetsar2/).It predicts the hepatotoxicity, carcinogenicity, nephrotoxicity, acute oral toxicity, non-target receptor binding, skin sensitization and ocular sensitivity (Yang et al., 2019).The preADMET (https://preadmet.webservice.bmdrc.org) analysis of 5g based on pharmacokinetics was also included to validate the predictions.Apart from these software, vNN ADMET (https://vnnadmet.bhsai.org/vnnadmet/login.xhtml) was used which utilizes the vNN approach (a set of 15 predictive models) for ADMET analysis.These models serve as rapid evaluators of crucial characteristics in drug candidates such as drug-related liver injury (Schyman et al., 2017).On the other hand, BioTransformer 3.0 (https://biotransformer.ca) represents a freely accessible online platform designed to integrate machine learning methodologies alongside rule-based systems.The primary objective of this tool is to forecast the metabolism of compounds within various tissues of humans, human gastrointestinal tract and soil and water microbiota from external environment (Wishart et al., 2022).XenoSite (https:// xenosite.org) is also an online tool that identifies actual atoms of a drug having probability of transformation during the metabolism in human body (Dang et al., 2020).

Molecular dynamics simulation
The best pose of the docked complex of 5g was studied using molecular dynamics (MD) analysis based on the docking results.The MD simulations were performed by the iMOD server (http://imods.chaconlab.org/)at 300 K constant temperature and 1 atm constant pressure (López-Blanco et al., 2014).The improved normal mode analysis (NMA) approach in inner coordinates has a user-friendly interface provided by the iMod server.All of the major browsers as well as contemporary mobile devices, are quite receptive and instantaneous with the online interface (Santra and Maiti, 2022).

Conclusion
In summary, the present work reports the design, synthesis and characterization of morpholine-thiophene hybrid thiosemicarbazones.The structures of these compounds were elucidated by FTIR, 1 H NMR, 13 C NMR spectroscopy and mass spectrometry.The synthetic compounds were tested against urease enzyme to combat ureolytic bacterial infections.The evaluation of these compounds against urease revealed a multi-fold superior inhibitory potential compared to the conventional inhibitor, thiourea.Among the tested compounds, 5g was recognized as the lead inhibitor, exhibiting remarkable inhibitory potential with an IC 50 value of 3.80 ± 1.9 µM.Enzyme's kinetics experiments revealed that the potent analogue exhibits uncompetitive inhibition, suggesting a unique mode of action against the urease enzyme.In docking analysis, 5g displayed a diverse variety of interactions with the active site amino acid residues of urease.In silico ADMET profile of 5g showed the druglikeness and leadlikeness properties with zero violations.Notably, the structural stability of the 5g complex was demonstrated through molecular dynamics simulations, further supporting its candidacy for preclinical studies.Altogether, the results achieved in the current research work contribute significantly towards the plethora of synthetic inhibitors and could potentially be targeted as a new template for the development of therapeutic candidates for ureolytic bacterial infections.

FIGURE 4
FIGURE 4Visual representation of HYDE of compound 5g showing the favorable atoms involved in affinity inside the active site of urease (PDB: 3LA4).

TABLE 2
The binding interactions between receptor residues and potent inhibitor 5g.

TABLE 3
ADME properties of compound 5g from SwissADME.